A focus of interest in this laboratory has been the study of genes that are involved in the formation of the neural crest and of its derivatives such as pigment cells, pharyngeal arches, and others. We found that the BTB domain-containing protein Kctd15 that is first expressed in the embryo at the neural plate border, may have a role in regulating the domain in which neural crest forms as overexpression of Kctd15 strongly inhibits neural crest specification. We have found that Kctd15 inhibits the activity of transcription factor AP-2, a regulatory molecule known to be critical for the specification of the neural crest in different animals. Mechanistic studies on the interaction between AP-2 and Kctd15 have been carried out. Ap-2 is a major regulator of neural crest formation, being involved in the initial induction of neural crest precursor cells as well as in later stages such as their migration and differentiation into multiple derivatives. Kctd15 is capable of binding AP-2 in co-immunoprecipitation experiments, and inhibits the activation of an AP-2 reporter both in cultured cells and in zebrafish embryos. In studying the mechanism of this inhibition we found that Kctd15 does not affect the level of AP-2 in the cell, does not inhibit dimerization of AP-2 which is required for activity, and does not affect nuclear localization of AP-2. Also, AP-2 remains bound to its cognate sites in chromatin in the presence of Kctd15. To probe the mechanism further we studied the activation domain of AP-2. Kctd15 binds to the activation domain, and specifically requires proline 59 for this interaction. An AP-2 mutant in which this proline is changed to alanine (P59A) is active in the reporter assay but cannot bind Kctd15, and its activity is not effectively inhibited by Kctd15. We conclude that Kctd15 inhibits AP-2 activity by specific binding to its activation domain which precludes its function. In further studies on Kctd15 we found that this protein is SUMOylated in vivo, both by SUMO1 and SUMO2/3. We identified the site of SUMO attachment and showed that mutation of the relevant lysine residue abolished SUMOylation. Non-SUMOylated Kctd15 was fully effective in inhibiting AP-2 function, but a stable fusion protein of Kctd15 and SUMO was not effective. We conclude that the non-SUMOylated form of Kctd15 functions in AP-2 inhibition, but leave open the possibility that the SUMOylated form has a role in a different biological function of Kctd15. To further analyze the function of Kctd15 in development we generated frameshift mutations in both Kctd15 paralogs with the use of the TALEN methodology. Homozygous Kctd15a and Kctd15b mutant fish are viable and fertile and show no overt phenotype. However, zebrafish mutant for both Kctd15a and Kctd15b, while viable and fertile, show a developmental delay and small size phenotype. These mutants also appear to have certain malformations in the craniofacial skeleton as well as some abnormalities in brain structure. The work on brain structure and function is a collaboration with the group of Harold Burgess in the Institute. We are currently studying the possibility that the small size phenotype may be due to inefficient feeding by the mutant fish. This might be due to the craniofacial abnormalities or to sensory deficits that potentially correlated with the observed changes in brain structure. In another approach at characterization of the Kctd15 mutant fish we entered a collaboration with the group of Anand Swaroop at NEI to compare the wild type and mutant transcriptomes by RNA-Seq. Sequencing was carried out at several stages of development, with results currently being analyzed. In our project on the function of E3 ubiquitin ligases in development we studied the role of the Lnx2 family in the initial specification of the pancreas in zebrafish. We found that the gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A TALEN induced frameshift null mutation in Lnx2a did not mimic this phenotype, being phenotypically normal. However, knockdown of the Lnx2b gene in the Lnx2a null mutant background mimicked the splice morpholino phenotype. Further and most compellingly, a mutation that removed the exon 2 splice donor site, which is targeted by the splice morpholino, effectively mimicked the exocrine suppression phenotype. From these findings we conclude that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. To analyze the mechanism of Lnx2 function in the pancreas in further detail we started from the earlier observation by others that human LNX1 destabilizes Numb. We could show that inhibition of Numb expression rescues the Lnx2a/b deficient phenotype, consistent with the hypothesis that Numb is a target for Lnx2 in the pancreas. Numb is a known inhibitor of the Notch signaling pathway, and Notch is known to have a critical role in pancreas differentiation. Further we found that inhibition of Lnx2a/b leads to a reduction in the number of Notch active cells in the pancreas. Thus we suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. An additional result of this study is to cast light on the complex relationships that may exist between genotype, phenotype, and morpholino effects. In the present example, disagreement between the consequences of a morpholino knockdown and a null mutation is not evidence of nonspecific effects of the morpholino but rather of a more complex relationship that could be revealed only by additional study. This conclusion is relevant to the ongoing discussion about the potential pitfalls of morpholino use as compared to the validity of conclusions based on morpholino reagents.